The present invention relates to a method and apparatus for performing a large number of reactions using array assembly. In particular, the present invention features a method and apparatus for performing a large number of chemical and biological reactions by bringing two arrays into close apposition and allowing reactants on the surfaces of two arrays to come into contact. The present invention is exemplified by performing a large number of polynucleotide amplification reactions using array assembly. In addition, the present invention features a method and apparatus for coupling the amplification of polynucleotides and the detection of sequence variations, expression levels, and functions thereof.
Intense efforts are under way to map and sequence the human genome and the genomes of many other species. In June 2000, the Human Genome Project and Celera Genomics announced that a rough draft of the human genome had been completed. This information, however, represents only a reference sequence of the 3-billion-base human genome. The remaining task lies in the determination of sequence variations (e.g., mutations, polymorphisms, haplotypes) and sequence functions, which are important for the study, diagnosis, and treatment of human genetic diseases.
In addition to the human genome, the mouse genome is being sequenced. Genbank provides about 1.2% of the 3-billion-base mouse genome and a rough draft of the mouse genome is expected to be available by 2003 and a finished genome by 2005. The Drosophila Genome Project has also been completed recently. Thus far, genomes of more than 30 organisms have been sequenced.
Traditional nucleic acid sequencing methods include the chemical cleavage method (or the Maxam-Gilbert method) and the chain termination method (or the Sanger method) (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The basic strategy for the chemical cleavage method is to specifically cleave the end-labeled DNA at only one type of nucleotide, which produces a set of labeled fragments. These labeled fragments are then separated according to their size by electrophoresis. The DNA sequence can be directly read off an autoradiogram. The chain termination method utilizes a DNA polymerase to make complementary copies of the single-stranded DNA being sequenced in the presence of a suitable primer and four deoxynucleoside triphosphates (dNTPs), of which at least one is labeled. In addition, a small amount of the 2xe2x80x2, 3xe2x80x2-dideoxynucleoside triphosphate of one of the bases is added to the sequencing reaction, which generates a series of truncated chains. Each truncated chain is terminated by the dideoxy analog at positions occupied by the corresponding base, because of the absence of a 3xe2x80x2-OH group. Electrophoresis separates these truncated chains according to their sizes, thus indicating the positions at which dideoxy incorporation occurs and in turn the corresponding normal nucleotide. Although the efficiency of the traditional sequencing methods has been improved by automation, the use of gel electrophoresis in both methods presents a limitation on the rate of sequencing.
While the traditional chemical cleavage and chain termination sequencing methods are capable of identifying the sequence of all nucleotides in a target nucleic acid, it is quite sufficient in many cases to know the sequence identity of a single nucleotide (or a few nucleotides) at a predetermined site, i.e., the detection of known sequence variations. During the past decade, the development of array-based hybridization technology has received great attention. This high throughput method, in which hundreds to thousands of polynucleotide probes immobilized on a solid surface are hybridized to target nucleic acids to gain sequence and function information, has brought economical incentives to many applications. See, e.g., McKenzie, S., et al., European Journal of Human Genetics 416-429 (1998), Green et al., Curr. Opin. in Chem. Biol. 2:404-410 (1998), Gerhold et al., TIBS, 24:168-173 (1999), Young, Cell 102:9-15 (2000), and U.S. Pat. Nos. 5,700,637, 6,054,270, 5,837,832, 5,744,305, and 5,445,943.
DNA array-based sequencing technology generally falls into two categories. The first category is sequencing by polynucleotide hybridization. Sets of polynucleotide probes, that differ by having A, T, C, or G substituted at or near the central position, are immobilized on a solid support by in situ synthesis or by deposition of pre-synthesized polynucleotide probes. Labeled target nucleic acids containing the sequences of interest will hybridize best to perfectly matched polynucleotide probes, whereas sequence variations will alter the hybridization pattern, thereby allowing the determination of mutations and polymorphic sites (Wang, D., et al., Science 280:1077-1082 (1998), Lipshutz, R., et al., Nature Genetics Supplement 21:20-24 (1999), and Drmanac et al., Nature Biotechnology 16:5-58 (1998)).
Alternatively, the de novo sequencing of target nucleic acids by polynucleotide hybridization may also be accomplished. For example, an array of all possible 8-mer polynucleotide probes may be hybridized with fluorescently labeled target nucleic acids, generating large amounts of overlapping hybridization data. The reassembling of this data by computer algorithm can determine the sequence of target nucleic acids. See, e.g., Drmanac, S. et al., Nature Biotechnology 116:54-58 (1998), Drmanac, S. et al. Genomics 4:114-28 (1989), and U.S. Pat. Nos. 5,202,231, 5,492,806, 5,525,464, 5,667,972, 5,695,940, 5,972,619, 6,018,041, and 6,025,136.
The second category is sequencing by primer extension reactions (also known as minisequencing). Typically, a DNA polymerase is used specifically to extend an interrogation primer, which anneals to the nucleic acids immediately 3xe2x80x2 of the single base substitution of interest, with a single labeled nucleoside triphosphate complementary to the single base substitution (Syvxc3xa4nen, Human Mutation 13:1-10 (1999), Syvxc3xa4nen et al., Genomics 8:684-692 (1990), Sokolov, Nucleic Acids Res. 18:3671 (1990), and Kuppuswami et al., Proc. Natl. Acad. Sci. USA 88:1143-1147 (1991)).
While methods of hybridization and primer extension-based nucleic acid sequencing have gained widespread acceptance in commercial areas, there are many limitations to the existing methods. The current methods for determining polynucleotide variations in a target nucleic acid employ discrete amplification steps and sequencing steps (Landegren et al., Genome Res. 8:769-776 (1998)). Thus, additional amount of time and labor is required to separate amplification products from the amplification primers and dNTPs before the sequencing reaction. Further, it is estimated that at least about 3,000,000 single nucleotide polymorphisms (SNPs) exist in an individual""s genome. As SNPs are dispersed throughout the genome, it is necessary to amplify a large number of discrete regions in the genome so that each SNP can be analyzed. Accordingly, the genetic analysis of a single individual""s SNPs can require more than 3,000,000 amplification reactions be carried out and the product of each amplification reaction be analyzed. In addition, genetic analysis of a disease may require extensive genotyping of hundreds of thousands of individuals. Therefore, the number of separate amplification and sequencing reactions can be in the millions. The cost in terms of time, labor, equipment, laboratory space and reagents for carrying out discrete amplification and sequencing reactions on a large-scale is prohibitively high. Finally, the designing, optimizing and manufacturing of probe-immobilized arrays can be costly as well. For example, photolithographic synthesis of an array with N-mer polynucleotides typically requires 4xc3x97N different chrome photolithographic masks (i.e., 100 different chrome masks for a 25-mer synthesis) (Singh-Gasson et al., Nature Biotechnol. 17:974-978 (1999)). This leads to high cost and long synthesis time. In addition, changing probe length and base composition in photolithographic DNA synthesis means changing masks, which again leads to high redesigning cost and long turnaround time for custom arrays. There is a need in micro array field to develop rapid and inexpensive methods for large-scale sequence variation and function analysis.
The present invention features novel applications of the array technology, in which large numbers of non-unimolecular reactions are initiated and performed by array assembly. This method is capable of generating large amounts of data or products per unit time by carrying out large numbers of reactions in parallel. Furthermore, the present invention is amendable to full automation.
The present invention relates to a method and apparatus for performing a large number of reactions using array assembly. In particular, the present invention features a method and apparatus for performing a large number of chemical and biological reactions by bringing two arrays into close apposition and allowing reactants on the surfaces of two arrays to come into contact. The present invention is exemplified by performing a large number of polynucleotide amplification reactions using array assembly. In addition, the present invention features a method and apparatus for coupling the amplification of polynucleotides and the detection of sequence variations, expression levels, and functions thereof.
Any suitable solid supports (also known as arrays, chips, etc.) may be used in the present invention. These materials include glass, silicon, quartz, nylon, polystyrene, polyethylene, polypropylene, polytetrafluorethylene, metal, among others. These materials typically have a rigid or semi-rigid surface. In some embodiments, at least one surface of the material is substantially flat. Typically, at least one solid support in the assembly is derivatized to provide covalent or noncovalent attachment to chemical or biological entities. Typically, the density of derivatized sites on an array is between about 10-10,000 per cm2, preferably below about 5,000, 1,000, 400, 200, 100, or 60 per cm2. The area of each site may be about 1xc3x9710xe2x88x923 to 5 mm2, preferably less than about 2, 1, 0.5, 0.2, or 0.1 mm2. Typically, the total number of derivatized sites on an array is between about 20-1,000,000, preferably, between about 20-500,000, 20-100,000, 20-50,000, 20-10,000, 20-5,000, 20-1,000, or 20-500. In some embodiments, an array may contain raised or depressed regions, e.g., features such as wells, raised regions, etched trenches, etc. The dimensions of these features are flexible, depending on factors, such as desirable reaction concentration, avoidance of air bubbles upon assembly, mechanical convenience and feasibility, etc. For example, the area of a well on an array is in general larger than one derivatized site on another array. The depth of a well may be less than about 1000 microns, preferably less than about 500, 200 or 100 microns. In preferred embodiments, surface tension arrays may be used. Certain reactants such as biopolymers (polynucleotides, polypeptides, etc.) are synthesized in situ to provide better yield and great flexibility.
The array assembly method provides an environment for simultaneously carrying out between about 10-500,000 reactions, preferably, above about 20, 50, 100, 200, 500, 1,000, 5,000, 10,000, 50,000, or 100,000 reactions. A myriad of chemical and biological reactions may be carried out using the instant method and apparatus. These reactions may involve cells, viruses, nucleic acids, proteins, carbohydrates, lipids, and small molecules, among others. In particular, a large number of polynucleotide amplification reactions or molecular binding reactions may be performed using array assembly. In some embodiments of the instant invention, one or more reactants may be immobilized on an array prior to array assembly. The immobilization may be covalent or non-covalent. For example, one or more reactants may be tethered to an immobilized moiety on the array. In certain embodiments of the instant invention, one or more reactants may be immobilized on an array via a releasable site, for example by tethering to an immobilized molecule with a releasable site. The immobilized reactants may be released from an array upon reacting with cleaving reagents prior to, during or after the array assembly. The release methods may include a variety of enzymatic, or non-enzymatic means, such as chemical, thermal, or photolytic treatment. In certain embodiments, detection of sequence variations or quantitation of polynucleotides may be coupled to the amplification reactions.
In one embodiment, the instant invention provides a system for performing a plurality of reactions comprising: a first solid support having a first reactant of each reaction confined to an area on the surface of said first solid support; and a second solid support having a second reactant of each reaction confined to an area on the surface of said second solid support; wherein said first and second solid supports are assembled to allow said first reactant of each reaction in contact with said second reactant of each reaction, thus providing an environment for performing said plurality of reactions in parallel.
The instant invention also provides a system for performing a plurality of reactions comprising: a first solid support having a reactant of each reaction immobilized on said first solid support; and a second solid support providing a chemical or mechanical separation of said plurality of reactions; wherein said first and second solid supports are assembled to provide an environment for performing said plurality of reactions in parallel.
The instant invention also provides a solid support for performing a plurality of polynucleotide amplification reactions wherein a releasable primer for each amplification reaction is immobilized on an area of the surface of said solid support.
The instant invention also provides a system for amplifying a plurality of target nucleic acids, comprising:
(a) a first solid support wherein
(1) the surface of said first solid support comprises a plurality of derivatized areas;
(2) a primer for each target nucleic acid or a sequence complementary to said primer is immobilized on a derivatized area of the first solid support; and
(b) a second solid support wherein said second solid support comprises a plurality of wells and each well corresponds to a primer for each target nucleic acid.
The instant invention also provides a system for amplifying a plurality of target nucleic acids, comprising:
(a) a first solid support wherein
(1) the surface of said first solid support comprises a plurality of derivatized areas;
(2) a forward primer for each target nucleic acid or a sequence complementary to said forward primer is immobilized on a derivatized area of the first solid support
(3) a reverse primer for each region of said target nucleic acid or a sequence complementary to said reverse primer is immobilized on another derivatized area of the first solid support;
(b) a second solid support wherein said second solid support comprises a plurality of wells and each well corresponds to the forward and reverse primers for each target nucleic acid.
The instant invention also provides a system for amplifying a plurality of target nucleic acids and detecting amplified products, comprising:
(a) a first solid support wherein
(1) the surface of said first solid support comprises a plurality of derivatized areas;
(2) a primer for each target nucleic acid or a sequence complementary to said primer is immobilized on a derivatized area of the first solid support;
(4) a probe comprising a subsequence, the same as or complementary to a subsequence of each target nucleic acid, is immobilized on another derivatized area of the first solid support;
(b) a second solid support wherein the surface of said second solid support comprises a plurality of reaction wells and each well corresponds the primer and the probe for each target nucleic acid.
In another embodiment, the instant invention provides a method for performing a plurality of reactions, comprising the steps of:
(a) obtaining a first solid support wherein a first reactant of each reaction is confined to an area on the surface of said first solid support;
(b) obtaining a second solid support wherein a second reactant of each reaction is confined to an area on the surface of said second solid support; and
(c) assembling said first and second solid support, wherein said first reactant on said first solid support is in contact with said second reactant on said second solid support, providing an environment for performing said plurality of reactions in parallel.
The instant invention also provides a method for performing a plurality of polynucleotide amplification reactions, comprising the steps of:
(a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;
(b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;
(c) assembling said first and second solid supports, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support; and
(d) releasing said primers.
The instant invention also provides a method for performing a plurality of polynucleotide amplification reactions and capturing amplification products, comprising the steps of:
(a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;
(b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;
(c) assembling said first and second solid supports, wherein said reactants of said polynucleotide aamplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;
(d) releasing said primers;
(e) generating amplification products of said polynucleotide amplification reactions; and
(f) capturing said amplified products by a plurality of immobilized polynucleotide probes on either said first or second solid support through hybridization.
The instant invention also provides a method for detecting a plurality of polynucleotide sequence variations, comprising the steps of:
(a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;
(b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;
(c) assembling said first and second solid support, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;
(d) releasing said primers;
(e) generating amplification products of said polynucleotide amplification reactions;
(f) capturing said amplified products by a plurality of immobilized polynucleotide probes on either said first or second array through hybridization; and
(g) detecting polynucleotide sequence variations by hybridization complexes in step (f).
The instant invention also provides a method for quantitating polynucleotides in a target nucleic acid, comprising the steps of:
(a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of finite areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;
(b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;
(c) assembling said first and second solid support, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;
(d) releasing said primers;
(e) generating amplification products of said polynucleotide amplification reactions; and
(f) quantitating amplified products.
The instant invention also provides a method for detecting polynucleotide sequence variations in a target nucleic acid, comprising the steps of:
(a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;
(b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each areas of said second solid support;
(c) assembling said first and second solid supports, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;
(d) releasing said primers;
(e) generating amplification products of said polynucleotide amplification reactions; and
(f) capturing said amplified products by a plurality of immobilized polynucleotide probes on either first or second array through hybridization; and
(g) detecting polynucleotide sequence variations by a polynucleotide modifying enzyme.
The instant invention also provides a method for amplifying a target nucleic acid, capturing the amplified product, and detecting a polynucleotide sequence variation in the amplified product, comprising the steps of:
(a) obtaining a first solid support wherein:
(1) the surface of said first solid support comprises a first, second, third, and fourth areas;
(2) a first chemical moiety, comprising a releasable forward primer for said target nucleic acid, is immobilized on said first area;
(3) a second chemical moiety, comprising a releasable reverse primer for said target nucleic acid, is immobilized on said second area;
(4) a first polynucleotide probe, comprising a subsequence complementary to one variant of said polynucleotide variation, is immobilized on said third area, said subsequence containing at least one interrogation position complementary to a corresponding nucleotide in said variant; and
(5) a second polynucleotide probe is immobilized to said fourth area, said second polynucleotide probe differing from said first polynucleotide probe by at least one nucleotide;
(b) obtaining a second solid support wherein the surface of said solid support comprises a reaction well and a mixture of reactants comprising a DNA polymerase, said target nucleic acid, and deoxynucleotides are placed within said reaction well;
(c) assembling said first and second solid support, wherein said mixture of reactants are in contact with said first, second, third, and fourth areas on said first solid support;
(d) releasing said releasable forward and reverse primers;
(e) generating the amplified product for said target nucleic acid;
(f) capturing the amplified product by said first or second polynucleotide probe through hybridization;
(g) disassembling said first and second solid supports;
(h) washing said first solid support;
(i) comparing the relative binding of two probes on said first solid support; and
(j ) identifying said polynucleotide variation in the amplified product.
The instant invention also provides a method for amplifying a target nucleic acid, capturing the amplified product, and detecting a polynucleotide sequence variation in the amplified product, comprising the steps of:
(a) obtaining a first solid support wherein:
(1) the surface of said first array comprises a first, second, third, and fourth areas;
(2) a first chemical moiety, comprising a releasable forward primer specific for said region of said target nucleic acid, is immobilized on said first area;
(3) a second chemical moiety, comprising a releasable reverse primer specific for said region of said target nucleic acid, is immobilized on said second area;
(4) a first polynucleotide probe, comprising a subsequence complementary to one variant of said polynucleotide variation, is immobilized on said third area, said subsequence containing at least one interrogation position complementary to a corresponding nucleotide in said variant; and
(5) a second polynucleotide probe is immobilized to said fourth area, said second probe differing from said first probe by at least one nucleotide;
(b) obtaining a second solid support wherein the surface of said solid support comprises a reaction well and a mixture of reactants comprising a DNA polymerase, said target nucleic acid, and deoxynucleotides are placed within said reaction well;
(c) assembling said first and second solid support, wherein said mixture of reactants are in contact with said first, second, third, and fourth areas on said first solid support;
(d) releasing said releasable forward and reverse primers;
(e) generating the amplified product for said target nucleic acid;
(f) capturing the amplified product by said first or second polynucleotide probes through hybridization;
(g) extending said one or more hybridization complexes in step (f);
(h) disassembling said first and second solid support;
(i) washing said first solid support; and
(j) identifying said polynucleotide variation using said one or more extended products in step (g).